EP4117703A1 - Verfahren und zusammensetzungen zur behandlung von coronavirus-infektionen - Google Patents

Verfahren und zusammensetzungen zur behandlung von coronavirus-infektionen

Info

Publication number
EP4117703A1
EP4117703A1 EP21715458.2A EP21715458A EP4117703A1 EP 4117703 A1 EP4117703 A1 EP 4117703A1 EP 21715458 A EP21715458 A EP 21715458A EP 4117703 A1 EP4117703 A1 EP 4117703A1
Authority
EP
European Patent Office
Prior art keywords
angiotensin
peptide
weight
ang
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP21715458.2A
Other languages
English (en)
French (fr)
Inventor
Richard Franklin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Constant Therapeutics LLC
Original Assignee
Constant Therapeutics LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Constant Therapeutics LLC filed Critical Constant Therapeutics LLC
Publication of EP4117703A1 publication Critical patent/EP4117703A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/085—Angiotensins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/64—Sulfonylureas, e.g. glibenclamide, tolbutamide, chlorpropamide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • Coronaviruses primarily target mucosal epithelia of respiratory and intestinal tracts resulting in respiratory and enteric symptoms.
  • the present invention provides, among other things, methods and compositions for treating individuals infected with a coronavirus, such that one or more symptoms or complications of the coronavirus infection is reduced in intensity, severity, duration, or frequency or has delayed onset.
  • angiotensin (1-7) peptides or angiotensin (1-7) receptor agonists can be used in treatment of lung disease or conditions, including bronchitis, pulmonary fibrosis, asthma, acute respiratory distress syndrome, and pneumonia that results as a complication of infection by viruses including coronaviruses COVID-19, SARS and MERS, respiratory syncytial virus, avian influenza A H5N1, H1N1 virus and others, as well as other pathogens.
  • the present application provides a method of treating a subject infected with a coronavirus comprising administering to the subject an angiotensin (1- 7) peptide or angiotensin (1-7) receptor agonist.
  • the subject infected with a coronavirus is infected with COVID-19, SARS or MERS.
  • the angiotensin (1-7) peptide is administered at an effective dose periodically at an administration interval such that one or more symptoms or complications of the coronavirus infection is reduced in intensity, severity, duration, or frequency or has delayed onset.
  • the one or more symptoms or complications of the coronavirus infection are selected from flu-like symptoms, fever, cough, myalgia, fatigue, pneumonia, or breathing difficulties. In some embodiments, the one or more symptoms or complications of the coronavirus infection comprise a cytokine storm.
  • the present application provides a method of treating a lung disease or condition comprising administering to the subject in need of treatment an angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist.
  • the lung disease or condition is selected from bronchitis, pneumonia, pulmonary fibrosis, asthma or acute respiratory distress syndrome.
  • the lung disease or condition is caused by a coronavirus infection.
  • the coronavirus infection is COVID-19, SARS or MERS.
  • the lung disease or condition is caused by avian flu H5N1.
  • the lung disease or condition is caused by H1N1.
  • the lung disease or condition is caused by RSV.
  • the administration is parenteral, rectal, oral, or a combination thereof.
  • the parenteral administration is intravenous, subcutaneous, inhalation, intradermal, transdermal, and/or transmucosal administration.
  • the administration is oral.
  • Ang-(1-7) is formulated with cyclodextrin for oral administration.
  • the angiotensin (1-7) peptide comprises an amino acid sequence of Asp 1 -Arg 2 -Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 (SEQ ID NO: 1) or a functional equivalent thereof.
  • the angiotensin (1-7) peptide is a linear peptide.
  • the angiotensin (1-7) peptide has SEQ ID NO: 1.
  • the angiotensin (1-7) peptide comprises an amino acid sequence of Asp 1 -Arg 2 -Val 3 -Ser 4 -Ile 5 -His 6 -Cys 7 (SEQ ID NO: 2) or a functional equivalent thereof.
  • the angiotensin (1-7) peptide has an amino acid sequence of Ala 1 -Arg 2 -Val 3 -Ser 4 -Ile 5 -His 6 -Cys 7 (SEQ ID NO: 3) or a functional equivalent thereof.
  • the angiotensin (1-7) peptide comprises one or more chemical modifications to increase protease resistance, serum stability and/or bioavailability.
  • the one or more chemical modifications comprise pegylation.
  • the angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist is administered in combination with another treatment of the coronavirus infection or another treatment of lung disease or condition.
  • the angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-1,000 ⁇ g/kg/day.
  • the angiotensin (l-7) receptor agonist is a compound with the following structure: or a pharmaceutically acceptable salt thereof.
  • animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development.
  • the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig).
  • animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms.
  • an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
  • biologically active refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
  • an agent that, when administered to an organism, has a biological effect on that organism is considered to be biologically active.
  • a peptide is biologically active
  • a portion of that peptide that shares at least one biological activity of the peptide is typically referred to as a “biologically active” portion.
  • a peptide has no intrinsic biological activity but that inhibits the effects of one or more naturally- occurring angiotensin compounds is considered to be biologically active.
  • Carrier or diluent refers to a pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) carrier or diluting substance useful for the preparation of a pharmaceutical formulation.
  • exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
  • Complication refers to an unfavorable evolution of a disease including the development of one or more signs, symptoms or, in some embodiments, even new pathological changes that manifest for a sustained period of time (e.g., weeks, months or years).
  • complication(s) may include a progression of a sign, symptom or other pathological change, for example, breathing difficulties growing worse over time, or a difficulty with one or more respiratory functions progressing to acute respiratory distress.
  • Dosage form As used herein, the terms “dosage form” and “unit dosage form” refer to a physically discrete unit of a therapeutic agent for the patient to be treated.
  • Dosing regimen is a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regime comprises a plurality of doses and at least two different time periods separating individual doses.
  • the therapeutic agent is administered continuously over a predetermined period. In some embodiments, the therapeutic agent is administered once a day (QD) or twice a day (BID).
  • QD once a day
  • BID twice a day
  • a functional derivative or equivalent may be a natural derivative or is prepared synthetically.
  • Exemplary functional derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved.
  • the substituting amino acid desirably has chemico-physical properties which are similar to that of the substituted amino acid. Desirable similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophilicity, and the like.
  • improve As used herein, the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein.
  • a “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • in vivo refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated.
  • isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure.
  • a substance is "pure” if it is substantially free of other components.
  • isolated cell refers to a cell not contained in a multi-cellular organism.
  • Polypeptide refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As is known to those skilled in the art, polypeptides may be processed and/or modified.
  • Protein The term “protein” as used herein refers to one or more polypeptides that function as a discrete unit.
  • a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit
  • the terms "polypeptide” and "protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” refers to the multiple polypeptides that are physically coupled and function together as the discrete unit.
  • Risk As will be understood from context, a "risk" of a disease, disorder, and/or condition comprises a likelihood that a particular individual will develop a disease, disorder, and/or condition (e.g.,lung injury). In some embodiments, risk is expressed as a percentage.
  • risk is from 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%.
  • risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples.
  • a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event (e.g., lung injury).
  • a reference sample or group of reference samples are from individuals comparable to a particular individual.
  • relative risk is 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • Stability refers to the ability of the therapeutic agent to maintain its therapeutic efficacy (e.g., all or the majority of its intended biological activity and/or physiochemical integrity) over extended periods of time.
  • the stability of a therapeutic agent, and the capability of the pharmaceutical composition to maintain stability of such therapeutic agent may be assessed over extended periods of time (e.g., for at least 1, 3, 6, 12, 18, 24, 30, 36 months or more).
  • pharmaceutical compositions described herein have been formulated such that they are capable of stabilizing, or alternatively slowing or preventing the degradation, of one or more therapeutic agents formulated therewith.
  • a stable formulation is one in which the therapeutic agent therein essentially retains its physical and/or chemical integrity and biological activity upon storage and during processes (such as freeze/thaw, mechanical mixing and lyophilization).
  • subject refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
  • a human includes pre- and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • subject is used herein interchangeably with “individual” or “patient.”
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • Substantially refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • Suffering from An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and/or condition.
  • Susceptible to An individual who is "susceptible to" a disease, disorder, and/or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, condition, or event may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, condition, and/or event (5) having undergone, planning to undergo, or requiring a transplant.
  • an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
  • therapeutically effective amount means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.
  • Treating refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
  • BRIEF DESCRIPTION OF THE DRAWING [0049] Drawings are for illustration purposes only; not for limitation.
  • Figure 1 shows an exemplary illustration of the renin-angiotensin (RAS) system.
  • RAS renin-angiotensin
  • Angiotensin Converting Enzyme cleaves Angiotensinogen (Ang) I to Angiotensinogen II which promotes lung injury through the AT1R signaling that leads to vasoconstriction and inflammation.
  • Angiotensin Converting Enzyme 2 cleaves Angiotensinogen II to Angiotensin (1-7) in the alternative renin- angiotensin pathway that exerts a protective effect against lung injury. Coronavirus infection downregulates ACE2 and promotes lung injury.
  • the present application provides a method of treating a subject infected with a coronavirus by administering to the subject an angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist.
  • the present invention further provides a method of treating a lung disease or condition, particularly caused by a virus infection, by administering to the subject in need of treatment an angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist.
  • Coronavirus Infection [0052] Coronaviruses (CoVs) are enveloped viruses with a positive-strand RNA genome, which primarily target respiratory and intestinal mucosal surfaces to establish respiratory and enteric infections.
  • Coronaviruses such as the human CoV isolates 229E and OC43 cause mild and self-limiting infections of the respiratory tract such as the common cold. Novel isolates HCoV-NL63 and HCoV-HKU1 have also been associated with common cold.
  • Some novel emerging coronaviruses have resulted in serious global outbreaks such as SARS in 2003, MERS in 2012 and COVID-19 in 2019. These diseases are associated with acute and often severe respiratory complications, especially in elderly and immunocompromised individuals.
  • COVID-19 infection results in respiratory symptoms like a runny nose, headache, dry cough, sore throat, shortness of breath, fever and fatigue.
  • COVID-19 Symptoms of COVID-19 may appear 2-14 days after exposure. Older adults and those with underlying health conditions and weakened immune systems may be at risk for more severe symptoms.
  • COVID-19 Once COVID-19 enters the human respiratory tract, it infects and propagates in cells lining the airway, causing damage that triggers local inflammation, recruiting immune cells in the vicinity to eradicate the virus. The immune response then recedes, and patients recover from mild disease. In elderly and other vulnerable individuals, there is instead an uncontrolled immune response, triggering an overproduction of immune cells and signaling molecules, leading to a cytokine storm often associated with a flood of immune cells into the lung. If an infection progresses to something more severe, it can cause bronchitis, pneumonia, kidney failure and even death.
  • Pneumonia associated with COVID-19 usually has bilateral lung involvement.
  • Pneumonia is characterized by inflammation of the alveoli or air sacs, which may fill with fluid or pus (purulent material), causing cough with phlegm or pus, fever, chills, and difficulty breathing.
  • Pneumonia may cause pleural effusion, i.e. fluid accumulation in the thin space between layers of tissue that line the lungs and chest cavity (pleura), which may lead to pus accumulation, infection and lung abscess.
  • pleural effusion i.e. fluid accumulation in the thin space between layers of tissue that line the lungs and chest cavity (pleura), which may lead to pus accumulation, infection and lung abscess.
  • pleura pleural effusion
  • lung fibrosis may occur. Pulmonary fibrosis occurs when the lung tissue around and between the alveoli becomes thickened, damaged and scarred, making it more difficult for oxygen to pass into the bloodstream.
  • ARDS acute respiratory distress syndrome
  • ALI acute lung injury
  • ARDS Predisposing factors for ARDS are diverse and include sepsis, aspiration, and pneumonias including infections with coronavirus, H1N1 and H5N1 avian influenza virus. Several other pathogens including Spanish flu virus and anthrax can result in ARDS. To date, no effective cure exists for ARDS.
  • SARS-CoV and COVID-19 rapidly spread person-to-person through respiratory secretions. Upon exposure of the host to the virus, the viral Spike protein (which projects through the viral envelope forming the characteristic spikes in the coronavirus), binds to epithelial cells expressing virus entry receptors, of which the angiotensin-converting enzyme 2 (ACE2) is one of the main receptors.
  • ACE2 angiotensin-converting enzyme 2
  • ACE2 In the respiratory tract, ACE2 is widely expressed on epithelial cells of alveoli, trachea, bronchi, bronchial serous glands, and alveolar monocytes and macrophages. The virus enters and replicates in these target cells, releasing mature virions which infect new target cells. Atypical pneumonia with rapid respiratory deterioration and lung injury can result due to increased levels of activated proinflammatory chemokines and cytokines.
  • Other pathogens including the influenza H5N1 and H1N1 viruses and RSV employ the ACE2 receptor for entry and the renin-angiotensin system plays a role in host response to infection.
  • Renin-Angiotensin System The renin-angiotensin system (RAS) maintains blood pressure and electrolyte balance in the body, important for cardiovascular and renal functions ( Figure 1). Renin is a protease that cleaves angiotensinogen to generate angiotensin (Ang) I.
  • Angiotensin- converting enzyme (ACE) cleaves Ang I to produce Ang II, which binds Ang II receptor type 1(AT1R) and Ang II receptor type 2 (AT2R) to carry out various biological functions in blood pressure regulation, mediating vasoconstriction, proliferative, fibrotic and inflammatory effects (Skeggs et al.1980; Corvol et al.1995).
  • ACE2 is a homologue of ACE that cleaves the octapeptide Ang II to a heptapeptide Ang-(1-7), which acts via the G-protein coupled Mas receptor in the alternative renin-angiotensin pathway to oppose the vasoconstrictive and inflammatory effects of the classical pathway mediated by ACE/AngII/AT1R signaling (Douglas et al., 2004, Kuba et al., 2013, Jiang et al, 2014).
  • Ang-(1-7) acts through a variety of downstream targets, including inhibition of fibrosis due to TGF- ⁇ , reduction of reactive oxygen species and oxidative stress, stimulation of brain derived neurotrophic factor (BDNF), activation of endothelial NO synthase (eNOS) and thus the release of NO, increasing vasodilation and endothelial protection.
  • BDNF brain derived neurotrophic factor
  • eNOS endothelial NO synthase
  • Ang-(1-7) functions in the stabilization of epithelial and endothelial membranes and stimulation of the proliferation and release of endothelial progenitor cells (EPCs) from the bone marrow increasing lung tissue repair and vascular remodeling.
  • EPCs endothelial progenitor cells
  • ACE2 functions to modulate innate immunity and influence the composition of gut microbiota.
  • Angiotensin II contributes to acute lung failure through stimulation of the AT1R, while ACE2 and AT2R negatively regulate this pathway and protect from acute lung failure.
  • ACE2 is found in alveolar epithelial cells, enterocytes in the small intestines and endothelial and smooth muscle cells in several organs.
  • ACE2 and its product Ang-(1-7) intrinsically protect lungs from severe acute injury by blocking the classical renin-angiotensin pathway.
  • ACE2 and Ang-(1-7) exert a protective role against a variety of pulmonary diseases, including but not limited to pulmonary fibrosis, acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, asthma, and chronic obstructive pulmonary disease.
  • this application provides a method of treating a subject infected with a coronavirus comprising administering to the subject an angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist.
  • angiotensin (1-7) peptide refers to both naturally-occurring Angiotensin (1-7) and any functional equivalent, analogue or derivative of naturally-occurring Angiotensin (1-7).
  • peptide and polypeptide are interchangeable terms and refer to two or more amino acids bound together by a peptide bond.
  • the terms “peptide” and “polypeptide” include both linear and cyclic peptide.
  • the terms “angiotensin-(1-7)”, “Angiotensin-(1-7)”, and “Ang-(1-7)” are used interchangeably.
  • Naturally-occurring Angiotensin (1-7) is a seven amino acid peptide shown below: Asp 1 -Arg 2 -Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 (SEQ ID NO: 1) It is part of the renin-angiotensin system and is converted from a precursor, also known as Angiotensinogen, which is an ⁇ -2-globulin that is produced constitutively and released into the circulation mainly by the liver. Angiotensinogen is a member of the serpin family and also known as renin substrate.
  • Human angiotensinogen is 452 amino acids long, but other species have angiotensinogen of varying sizes. Typically, the first 12 amino acids are the most important for angiotensin activity: Asp 1 -Arg 2 -Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 -Phe 8 -His 9 -Leu 10 -Val 11 -Ile 12 (SEQ ID NO: 3) [0067] Different types of angiotensin may be formed by the action of various enzymes. For example, Angiotensin (1-7) is generated by action of Angiotensin- converting enzyme 2 (ACE 2). [0068] Ang-(1-7) is an endogenous ligand for Mas receptors.
  • Mas receptors are G- protein coupled receptor containing seven transmembrane spanning regions.
  • angiotensin-(1-7) receptor encompasses the G Protein-Coupled Mas Receptors.
  • naturally-occurring Angiotensin (1-7) includes any Angiotensin (1-7) peptide purified from natural sources and any recombinantly produced or chemically synthesized peptides that have an amino acid sequence identical to that of the naturally-occurring Angiotensin (1-7).
  • an angiotensin (1-7) peptide suitable for the present invention is a functional equivalent of naturally-occurring Ang-(1-7).
  • a functional equivalent of naturally-occurring Ang-(1-7) refers to any peptide that shares amino acid sequence identity to the naturally-occurring Ang-(1-7) and retain substantially the same or similar activity as the naturally-occurring Ang-(1-7).
  • a functional equivalent of naturally-occurring Ang-(1-7) described herein has pro-angiogenic activity as determined using methods described herein or known in the art, or an activity such as nitric oxide release, vasodilation, improved endothelial function, antidiuresis, or one of the other properties discussed herein, that positively impacts angiogenesis.
  • a functional equivalent of naturally-occurring Ang-(1-7) described herein can bind to or activate an angiotensin-(1- 7) receptor (e.g., the G protein-coupled Mas receptor) as determined using various assays described herein or known in the art.
  • Ang-(1-7) inhibits fibrosis by downregulating TGF- ⁇ . In some embodiments, Ang-(1-7) reduces reactive oxygen species and oxidative stress. In some embodiments, Ang-(1-7) stimulates BDNF. In some embodiments, a functional equivalent of Ang-(1-7) is also referred to as an angiotensin (1-7) analogue or derivative, or functional derivative. [0071] Typically, a functional equivalent of angiotensin (1-7) shares amino acid sequence similarity to the naturally-occurring Ang-(1-7).
  • a functional equivalent of Ang-(1-7) contains a sequence that includes at least 3 (e.g., at least 4, at least 5, at least 6, at least 7) amino acids from the seven amino acids that appear in the naturally-occurring Ang-(1-7), wherein the at least 3 (e.g., at least 4, at least 5, at least 6, or at least 7) amino acids maintain their relative positions and/or spacing as they appear in the naturally-occurring Ang- (1- 7).
  • a functional equivalent of Ang-(1-7) also encompasses any peptide that contains a sequence at least 50% (e.g., at least 60%, 70%, 80%, or 90%) identical to the amino acid sequence of naturally-occurring Ang- (1-7). Percentage of amino acid sequence identity can be determined by alignment of amino acid sequences. Alignment of amino acid sequences can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul et al., Methods in Enzymology 266, 460-480 (1996); http://blast.wustl/edu/blast/README.html). WU-BLAST-2 uses several search parameters, most of which are set to the default values.
  • HSP score (S) and HSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the minimum values may be adjusted and are set as indicated above.
  • a functional equivalent, analogue or derivative of Ang-(1-7) is a fragment of the naturally-occurring Ang-(1-7).
  • a functional equivalent, analogue or derivative of Ang-(1-7) contains amino acid substitutions, deletions and/or insertions in the naturally-occurring Ang-(1- 7).
  • Ang-(1-7) functional equivalents, analogues or derivatives can be made by altering the amino acid sequences by substitutions, additions, and/or deletions.
  • one or more amino acid residues within the sequence of the naturally-occurring Ang- (1- 7) can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration.
  • substitution for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the positively charged (basic) amino acids include arginine, lysine, and histidine.
  • the nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan, and methionine.
  • the uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the negatively charged (acid) amino acids include glutamic acid and aspartic acid.
  • the amino acid glycine may be included in either the nonpolar amino acid family or the uncharged (neutral) polar amino acid family. Substitutions made within a family of amino acids are generally understood to be conservative substitutions. For example, the amino acid sequence of a peptide inhibitor can be modified or substituted.
  • Examples of Ang-(1-7) functional equivalents, analogues and derivatives are described in the section entitled "Exemplary Angiotensin (l -7) Peptides" below.
  • An angiotensin-(1- 7) peptide can be of any length.
  • an angiotensin-(1- 7) peptide according to the present invention can contain, for example, from 4-25 amino acids (e.g., 4-20, 4-15, 4-14, 4-13, 4-12, 4-11, 4- 10, 4-9, 4-8, 4-7 amino acids).
  • the linear peptide contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.
  • an angiotensin-( 1-7) peptide contains one or more modifications to increase protease resistance, serum stability and/or bioavailability.
  • suitable modifications are selected from pegylation, acetylation, glycosylation, biotinylation, substitution with D-amino acid and/or un-natural amino acid, and/or cyclization of the peptide.
  • amino acid in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain.
  • an amino acid has the general structure H2N-C(H)(R)-COOH.
  • an amino acid is a naturally-occurring amino acid.
  • an amino acid is a synthetic or un-natural amino acid (e.g., a,a-disubstituted amino acids, N-alkyl amino acids); in some embodiments, an amino acid is a d-amino acid; in certain embodiments, an amino acid is an 1-amino acid.
  • Standard amino acid refers to any of the twenty standard amino acids commonly found in naturally occurring peptides including both 1- and d- amino acids which are both incorporated in peptides in nature.
  • “Nonstandard” or “unconventional amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • “synthetic or un- natural amino acid” encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions.
  • Amino acids including carboxy- and/or amino- terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, and/or substitution with other chemical groups that can change the peptide's circulating half-life without adversely affecting its activity.
  • Examples of unconventional or un- natural amino acids include, but are not limited to, citrulline, omithine, norleucine, norvaline, 4-(E)-butenyl-4(R)-methyl-N-methylthreonine (MeBmt), N-methyl- leucine (MeLeu), aminoisobutyric acid, statine, and N-methyl-alanine (MeAla).
  • Amino acids may participate in a disulfide bond.
  • amino acid is used interchangeably with "amino acid residue,” and may refer to a free amino acid and/or to an amino acid residue of a peptide.
  • angiotensin-( 1-7) peptides contain one or more L-amino acids, D-amino acids, and/or un-natural amino acids.
  • peptidomimetics or peptide analogs are also encompassed by the present invention. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. The non-peptide compounds are termed “peptide mimetics” or peptidomimetics (Fauchere et al., Infect.
  • paradigm polypeptide i.e., a polypeptide that has a biological or pharmacological activity
  • Such peptide mimetics may have significant advantages over naturally- occurring polypeptides including more economical production, greater chemical stability, enhanced pharmacological properties (e.g., half-life, absorption, potency, efficiency, etc.), reduced antigenicity and others.
  • Ang-(1-7) peptides also include other types of peptide derivatives containing additional chemical moieties not normally part of the peptide, provided that the derivative retains the desired functional activity of the peptide.
  • examples of such derivatives include (1) N-acyl derivatives of the amino terminal or of another free amino group, wherein the acyl group may be an alkanoyl group (e.g., acetyl, hexanoyl, octanoyl) an aroyl group (e.g., benzoyl) or a blocking group such as F-moc (fluorenylmethyl-0-CO-); (2) esters of the carboxy terminal or of another free carboxy or hydroxyl group; (3) amide of the carboxy-terminal or of another free carboxyl group produced by reaction with ammonia or with a suitable amine; (4) phosphorylated derivatives; (5) derivatives conjugated to an antibody or other biological ligand and other types of derivatives; and (6) derivative
  • Ang-(1-7) peptides may be obtained by any method of peptide synthesis known to those skilled in the art, including synthetic (e.g., exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, classical solution synthesis, native-chemical ligation) and recombinant techniques.
  • the peptides or peptides derivatives can be obtained by solid phase peptide synthesis, which in brief, consist of coupling the carboxyl group of the C- terminal amino acid to a resin (e.g., benzhydrylamine resin, chloromethylated resin, hydroxymethyl resin) and successively adding N-alpha protected amino acids.
  • the protecting groups may be any such groups known in the art.
  • alpha amino protecting groups include acyl type protecting groups (e.g., trifluoroacetyl, formyl, acetyl), aliphatic urethane protecting groups (e.g., t-butyloxycarbonyl (BOC), cyclohexyloxycarbonyl), aromatic urethane type protecting groups (e.g., fluorenyl-9- methoxy-carbonyl (Fmoc), benzyloxycarbonyl (Cbz), Cbz derivatives) and alkyl type protecting groups (e.g., triphenyl methyl, benzyl).
  • acyl type protecting groups e.g., trifluoroacetyl, formyl, acetyl
  • aliphatic urethane protecting groups e.g., t-butyloxycarbonyl (BOC), cyclohexyloxycarbonyl
  • aromatic urethane type protecting groups e.g., fluorenyl-9
  • the amino acids side chain protecting groups include benzyl (for Thr and Ser), Cbz (Tyr, Thr, Ser, Arg, Lys), methyl ethyl, cyclohexyl (Asp, His), Boe (Arg, His, Cys) etc.
  • the protecting groups may be removed at a convenient subsequent stage using methods known in the art.
  • Ang-(1-7) peptides may be synthesized according to the FMOC protocol in an organic phase with protective groups. Desirably, the peptides are purified with a yield of 70% with high-pressure liquid chromatography (HPLC) on a C 18 chromatography column and eluted with an acetonitrile gradient of 10-60%.
  • HPLC high-pressure liquid chromatography
  • Ang-(1-7) peptides may be prepared in recombinant systems using, for example, polynucleotide sequences encoding the polypeptides. It is understood that a polypeptide may contain more than one of the above-described modifications within the same polypeptide.
  • peptides may be effective in eliciting a biological activity in vitro, their effectiveness in vivo might be reduced by the presence of proteases.
  • Serum proteases have specific substrate requirements.
  • the substrate must have both L-amino acids and peptide bonds for cleavage.
  • exopeptidases which represent the most prominent component of the protease activity in serum, usually act on the first peptide bond of the peptide and require a free N-terminus (Powell et al., Pharm. Res. 10:1268-1273 (1993)).
  • modified versions of peptides retain the structural characteristics of the original L- amino acid peptides that confer the desired biological activity of Ang-(1-7) but are advantageously not readily susceptible to cleavage by protease and/or exopeptidases.
  • a peptide derivative or peptidomimetic of the present invention may be all L, all D or mixed D, L peptide, in either forward or reverse order.
  • the presence of an N-terminal or C-terminal D-amino acid increases the in vivo stability of a peptide since peptidases cannot utilize a D-amino acid as a substrate (Powell et al., Pharm. Res.10:1268-1273 (1993)).
  • Reverse- D peptides are peptides containing D-amino acids, arranged in a reverse sequence relative to a peptide containing L-amino acids.
  • the C-terminal residue of an L-amino acid peptide becomes N-terminal for the D-amino acid peptide, and so forth.
  • Reverse D-peptides retain the same secondary conformation and therefore similar activity, as the L-amino acid peptides, but are more resistant to enzymatic degradation in vitro and in vivo, and thus can have greater therapeutic efficacy than the original peptide (Brady and Dodson, Nature 368:692-693 (1994); Jameson et al., Nature 368:744-746 (1994)).
  • a reverse-L peptide may be generated using standard methods where the C-terminus of the parent peptide becomes takes the place of the N-terminus of the reverse-L peptide. It is contemplated that reverse L-peptides of L-amino acid peptides that do not have significant secondary structure (e.g., short peptides) retain the same spacing and conformation of the side chains of the L-amino acid peptide and therefore often have the similar activity as the original L-amino acid peptide. Moreover, a reverse peptide may contain a combination of L- and D-amino acids. The spacing between amino acids and the conformation of the side chains may be retained resulting in similar activity as the original L- amino acid peptide.
  • Another effective approach to confer resistance to peptidases acting on the N- terminal or C-terminal residues of a peptide is to add chemical groups at the peptide termini, such that the modified peptide is no longer a substrate for the peptidase.
  • One such chemical modification is glycosylation of the peptides at either or both termini.
  • Certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of peptides in human serum (Powell et al., Pharm. Res.10:1268-1273 (1993)).
  • N-terminal alkyl group consisting of a lower alkyl of from one to twenty carbons, such as an acetyl group, and/or the addition of a C-terminal amide or substituted amide group.
  • the present invention includes modified peptides consisting of peptides bearing an N- terminal acetyl group and/or a C-terminal amide group. [0089] Substitution of non-naturally-occurring amino acids for natural amino acids in a subsequence of the peptides can also confer resistance to proteolysis.
  • Such a substitution can, for instance, confer resistance to proteolysis by exopeptidases acting on the N-terminus without affecting biological activity.
  • non-naturally- occurring amino acids include ⁇ , ⁇ -disubstituted amino acids, N-alkyl amino acids, C- ⁇ -methyl amino acids, ⁇ -amino acids, and ⁇ -methyl amino acids.
  • Amino acids analogs useful in the present invention may include, but are not limited to, ⁇ -alanine, norvaline, norleucine, 4-aminobutyric acid, orithine, hydroxyproline, sarcosine, citrulline, cysteic acid, cyclohexylalanine, 2-aminoisobutyric acid, 6-aminohexanoic acid, t-butylglycine, phenylglycine, o-phosphoserine, N-acetyl serine, N- formylmethionine, 3- methylhistidine and other unconventional amino acids. Furthermore, the synthesis of peptides with non-naturally-occurring amino acids is routine in the art.
  • constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods well known in the art (Rizo and Gierasch, Ann. Rev. Biochem.61:387-418 (1992)).
  • constrained peptides may be generated by adding cysteine residues capable of forming disulfide bridges and, thereby, resulting in a cyclic peptide.
  • Cyclic peptides can be constructed to have no free N- or C-termini. Accordingly, they are not susceptible to proteolysis by exopeptidases, although they may be susceptible to endopeptidases, which do not cleave at peptide termini.
  • amino acid sequences of the peptides with N-terminal or C-terminal D-amino acids and of the cyclic peptides are usually identical to the sequences of the peptides to which they correspond, except for the presence of N- terminal or C-terminal D-amino acid residue, or their circular structure, respectively.
  • Cyclic Peptides [0091]
  • a functional equivalent, analogue or derivative of naturally-occurring Ang-(1-7) is a cyclic peptide.
  • a cyclic peptide has an intramolecular covalent bond between two non-adjacent residues.
  • the intramolecular bond may be a backbone to backbone, side-chain to backbone or side- chain to side-chain bond (i.e., terminal functional groups of a linear peptide and/or side-chain functional groups of a terminal or interior residue may be linked to achieve cyclization).
  • Typical intramolecular bonds include disulfide, amide and thioether bonds.
  • a variety of means for cyclizing polypeptides are well known in the art, as are many other modifications that can be made to such peptides. For a general discussion, see International Patent Publication Nos. WO 01/53331 and WO 98/02452, the contents of which are incorporated herein by reference.
  • Cyclic peptides as described herein may comprise residues of L-amino acids, D-amino acids, or any combination thereof.
  • Amino acids may be from natural or non-natural sources, provided that at least one amino group and at least one carboxyl group are present in the molecule; ⁇ - and ⁇ -amino acids are generally preferred.
  • Cyclic peptides may also contain one or more rare amino acids (such as 4-hydroxyproline or hydroxylysine ), organic acids or amides and/or derivatives of common amino acids, such as amino acids having the C-terminal carboxylate esterified (e.g., benzyl, methyl or ethyl ester) or amidated and/or having modifications of the N-terminal amino group (e.g., acetylation or alkoxycarbonylation), with or without any of a wide variety of side-chain modifications and/or substitutions (e.g., methylation, benzylation, t-butylation, tosylation, alkoxycarbonylation, and the like).
  • rare amino acids such as 4-hydroxyproline or hydroxylysine
  • organic acids or amides and/or derivatives of common amino acids such as amino acids having the C-terminal carboxylate esterified (e.g., benzyl, methyl or ethyl ester) or amidated and/
  • Suitable derivatives include amino acids having an N-acetyl group (such that the amino group that represents the N- terminus of the linear peptide prior to cyclization is acetylated) and/or a C-terminal amide group (i.e., the carboxy terminus of the linear peptide prior to cyclization is amidated).
  • Residues other than common amino acids that may be present with a cyclic peptide include, but are not limited to, penicillamine, ⁇ , ⁇ -tetramethylene cysteine, ⁇ , ⁇ -pentamethylene cysteine, ⁇ -mercaptopropionic acid, ⁇ -pentamethylene- mercaptopropionic acid, 2- mercaptobenzene, 2- mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid, ⁇ -aminoadipic acid, m-aminomethylbenzoic acid and ⁇ , ⁇ -diaminopropionic acid.
  • cyclization may be achieved by any of a variety of techniques well known in the art.
  • a bond may be generated between reactive amino acid side chains.
  • a disulfide bridge may be formed from a linear peptide comprising two thiol-containing residues by oxidizing the peptide using any of a variety of methods.
  • air oxidation of thiols can generate disulfide linkages over a period of several days using either basic or neutral aqueous media.
  • the peptide is used in high dilution to minimize aggregation and intermolecular side reactions.
  • cyclization may be achieved by amide bond formation.
  • a peptide bond may be formed between terminal functional groups (i.e., the amino and carboxy termini of a linear peptide prior to cyclization).
  • the linear peptide comprises a D-amino acid.
  • cyclization may be accomplished by linking one terminus and a residue side chain or using two side chains, with or without an N-terminal acetyl group and/or a C-terminal amide.
  • Residues capable of forming a lactam bond include lysine, ornithine (Orn), ⁇ -amino adipic acid, m-aminomethylbenzoic acid, ⁇ , ⁇ -diaminopropionic acid, glutamate or aspartate. Methods for forming amide bonds are generally well known in the art.
  • carbodiimide-mediated lactam formation can be accomplished by reaction of the carboxylic acid with DCC, DIC, ED AC or DCCI, resulting in the formation of an O- acylurea that can be reacted immediately with the free amino group to complete the cyclization.
  • cyclization can be performed using the azide method, in which a reactive azide intermediate is generated from an alkyl ester via a hydrazide.
  • cyclization can be accomplished using activated esters. The presence of electron withdrawing substituents on the alkoxy carbon of esters increases their susceptibility to aminolysis.
  • a thioether linkage may be formed between the side chain of a thiol-containing residue and an appropriately derivatized a-amino acid.
  • a lysine side chain can be coupled to bromoacetic acid through the carbodiimide coupling method (DCC, EDAC) and then reacted with the side chain of any of the thiol containing residues mentioned above to form a thioether linkage.
  • any two thiol containing side- chains can be reacted with dibromoethane and diisopropylamine in DMF.
  • Exemplary Angiotensin-(1-7) Peptides [0094] In certain aspects, the invention provides linear angiotensin-(1- 7) peptides.
  • Ang-(1-7) As discussed above, the structure of naturally-occurring Ang-(1-7) is as follows: Asp 1 -Arg 2 -Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 (SEQ ID NO: 1) [0095]
  • the peptides and peptide analogs of the invention can be generally represented by the following sequence: Xaa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Xaa 5 -Xaa 6 -Xaa 7 (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof.
  • Xaa 1 is any amino acid or a dicarboxylic acid.
  • Xaa 1 is Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me 2 Gly (N, N- dimethylglycine), Pro, Bet (betaine, 1-carboxy-N,N,N-trimethylmethanaminium hydroxide), Glu, Gly, Asp, Sar (sarcosine) or Suc (succinic acid).
  • Xaa 1 is a negatively-charged amino acid, such as Asp or Glu, typically Asp.
  • Xaa 2 is Arg, Lys, Ala, Cit (citrulline), Orn (omithine), acetylated Ser, Sar, D-Arg and D- Lys.
  • Xaa2 is a positively-charged amino acid such as Arg or Lys, typically Arg.
  • Xaa 3 is Val, Ala, Leu, Nle (norleucine), Ile, Gly, Lys, Pro, HydroxyPro (hydroxyproline), Aib (2-aminoisobutyric acid), Acpc or Tyr.
  • Xaa 3 is an aliphatic amino acid such as Val, Leu, Ile or Nle, typically Val or Nle.
  • Xaa 4 is Tyr, Tyr(PO3 ), Thr, Ser, homoSer (homoserine), azaTyr (aza- ⁇ 1 -homo-L- tyrosine) or Ala.
  • Xaa 4 is a hydroxyl-substituted amino acid such as Tyr, Ser or Thr, typically Tyr.
  • Xaa 5 is Ile, Ala, Leu, norLeu, Val or Gly.
  • Xaa 5 is an aliphatic amino acid such as Val, Leu, Ile or Nle, typically Ile.
  • Xaa 6 is His, Arg or 6-NH2-Phe (6-aminophenylalaine).
  • Xaa 6 is a fully or partially positively-charged amino acid such as Arg or His.
  • Xaa 7 is Cys, Pro or Ala.
  • one or more of Xaa 1 -Xaa 7 is identical to the corresponding amino acid in naturally-occurring Ang-(1- 7).
  • all but one or two of Xaa 1 -Xaa 7 are identical to the corresponding amino acid in naturally-occurring Ang-(1-7).
  • all of Xaa 1 -Xaa 6 are identical to the corresponding amino acid in naturally-occurring Ang-(1-7).
  • Xaa 3 is Nle.
  • Xaa 3 When Xaa 3 is Nle, one or more of Xaa 1 -Xaa 2 and Xaa 4-7 are optionally identical to the corresponding amino acid in naturally-occurring Ang-(1-7). In certain such embodiments, all but one or two of Xaa 1 -Xaa 2 and Xaa 4-7 are identical to the corresponding amino acid in naturally- occurring Ang-(1-7).
  • all of Xaa 1 -Xaa 2 and Xaa 4-7 are identical to the corresponding amino acid in naturally-occurring Ang-(1-7), resulting in the amino acid sequence: Asp 1 -Arg 2 -Nle 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 (SEQ ID NO: 5).
  • the peptide has the amino acid sequence Asp 1 -Arg 2 -Val 3 -Ser 4 -Ile 5 -His 6 -Cys 7 (SEQ ID NO: 6) or Asp 1 -Arg 2 -Val 3 -ser o -Ile 5 - His 6 -Cys 7 (SEQ ID NO: 2).
  • Exemplary Cyclic Angiotensin (1-7) Peptides [0099]
  • the invention provides a cyclic angiotensin-(1-7) (Ang-(1-7)) peptide analog comprising a linkage, such as between the side chains of amino acids corresponding to positions Tyr 4 and Pro 7 in Ang.
  • peptide analogs typically comprise 7 amino acid residues, but can also include a cleavable sequence.
  • the invention includes fragments and analogs where one or more amino acids are substituted by another amino acid (including fragments).
  • One example of such an analog is Asp 1 -Arg 2 -Val 3 -Ser 4 -Ile 5 -His 6 -Cys 7 (SEQ ID NO: 22), wherein a linkage is formed between Ser 4 and Cys 7 .
  • a thioether bridge is also referred to as a monosulfide bridge or, in the case of Ala-S-Ala, as a lanthionine bridge.
  • Thioether bridge-containing peptides can be formed by two amino acids having one of the following formulas: [0101]
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, an alkyl (e.g., C1-C6 alkyl, C1-C4 alkyl) or an aralkyl group, where the alkyl and aralkyl groups are optionally substituted with one or more halogen, -OH or -NRR' groups (where R and R' are independently –H or C1-C4 alkyl).
  • R1 R2, R3, R4, R5 and R6 are each independently –H or -CH3, such where all are –H.
  • the invention provides an Ang analog or derivative comprising a thioether bridge according to formula (I).
  • R 1 , R 2 , R 3 and R 4 are independently selected from -H and -CH3 .
  • Peptides comprising a thioether bridge according to formula (I) can be produced, for example, by lantibiotic enzymes or by sulfur extrusion of a disulfide.
  • the disulfide from which the sulfur is extruded can be formed by D- cysteine in position 4 and L-cysteine in position 7 or by D- cysteine in position 4 and L-penicillamine in position 7 (see, e.g., Gaisme, Trent and Spatola (2003) Biopolymers 71, 534- 551).
  • the linkage of the two amino acids can be the bridges depicted in Formula (II) or Formula (III).
  • Peptides comprising a thioether bridge according to Formula (II) can be made, for example, by sulfur extrusion of a disulfide formed by D- homocysteine in position 4 and L-cysteine in position 7.
  • peptides comprising a thioether bridge as in Formula (III) can be made, for example, by sulfur extrusion of a disulfide formed by D-cysteine in position 4 and L- homocysteine in position 7.
  • the Ang analogs and derivatives of the invention vary in length and amino acid composition.
  • the Ang analogs and derivatives of the invention preferably have biological activity or are an inactive precursor molecule that can be proteolytically activated (such as how angiotensin(I), with 10 amino acids, is converted to active fragments by cleavage of 2 amino acids).
  • an Ang analog or derivative can vary but is typically between from about 5 to 10 amino acids, as long as the “core” pentameric segment comprising the 3-7 Nle-thioether-ring structure is encompassed.
  • the amino acid sequence of an analog or derivative of the invention can vary, typically provided that it is biologically active or can become proteolytically activated.
  • Biological activity of an analog or derivative can be determined using methods known in the art, including radioligand binding studies, in vitro cell activation assays and in vivo experiments. See, for example, Godeny and Sayeski, (2006) Am. J Physiol. Cell. Physiol. 291 :C1297-1307; Sarr et al., Cardiovasc. Res.
  • Ang analogs and derivatives where only the length of the peptide is varied include the following: a 4,7-cyclized analog designated [Cyc 4 7 ]Ang --(1-7), which is derived from natural Ang- (1-7) (Asp 1 -Arg 2 -Vai3-Cyc 4 -Ile 5 -His 6 -Cyc 7 , SEQ ID NO:7).
  • These analogs can have one of the thioether bridges shown in Formulae (I)-(III) as the - Cyc 47 moiety, for example, where Cyc 4 and Cyc 7 are represented by Formula (I), such as where R 1 -R 4 are each -H or -CH3, typically -H.
  • Cyc 4 and Cyc 7 are represented by Formula (I), such as where R 1 -R 4 are each -H or -CH3, typically -H.
  • the amino acids at positions 4 and 7 of the Cyc4-7-analog are modified to allow introduction of the thioether-ring structures shown above.
  • the amino acids at positions other than 3, 4 and 7 can be the same or different from the naturally-occurring peptide, typically provided that the analog retains a biological function.
  • biological function refers to one or both of an analog's susceptibility to angiotensin-converting enzymes that can cleave it to a biologically active fragment (e.g. Ang-(1-8) or Ang-(1-7)) or the biological activity of the fragment itself.
  • an Ang analog or derivative of the invention has no intrinsic function but inhibits the effects of one or more naturally-occurring angiotensin compounds.
  • an Ang analog of the invention is represented by Formula (IV): Xaa 1 -Xaa 2 -Xaa 3 -Cyc 4 -Xaa 5 -Xaa 6 -Cyc 7 (IV, SEQ ID NO: 14)
  • Xaa 1 is any amino acid, but typically a negatively-charged amino acid such as Glu or Asp, more typically Asp.
  • Xaa 2 is a positively-charged amino acid such as Arg or Lys, typically Arg.
  • Xaa 3 is an aliphatic amino acid, such as Leu, Ile or Val, typically Val. Cyc 4 forms a thioether bridge in conjunction with Cyc 7 .
  • Cyc 4 can be a D- stereoisomer and/or a L-stereoisomer, typically a D-stereoisomer.
  • Examples of Cyc 4 (taken with Cyc 7 ) are shown in Formulas (I), (II) and (III).
  • the R groups in Formulae (I), (II) and (III) are -H or -CH3, especially -H.
  • Xaa 5 is an aliphatic amino acid, such as Leu, Ile or Val, typically Ile.
  • Xaa 6 is His.
  • Cyc 7 forms a thioether bridge in conjunction with Cyc 4 , such as in Formula (I), (II) or (III).
  • Cyc 7 can be a D-stereoisomer and/or a L-stereoisomer, typically a L-stereoisomer.
  • Examples of Cyc 7 (taken with Cyc 4 ) are shown in Formulas (I), (II), (III) and (IV).
  • the R groups in Formulae (I), (II),) and (III) and (IV) are -H or -CH3, especially -H.
  • one or more of Xaa 1 -Xaa 6 (excluding Cyc 4 and Cyc 7 ) is identical to the corresponding amino acid in naturally-occurring Ang-(1- 7).
  • Xaa 1 - Xaa 6 are identical to the corresponding amino acid in naturally-occurring Ang-(1-7). In other embodiments, all of Xaa 1 - Xaa 6 are identical to the corresponding amino acid in naturally-occurring Ang- (1-7).
  • Cyc 4 and Cyc 7 are independently selected from , Abu (2- aminobutyric acid) and Ala (alanine), where Ala is present in at least one position.
  • cyclic analogs can have a thioether linkage formed by -Ala 4 -S-Ala 7 - (Formula (I), where R 1 -R 4 are each -H); -Ala 4 -S-Abu 7 - (Formula (I): R 1 -R 3 are -H and R 4 is -CH3 ) or -Abu 4 -S-Ala 7 - (Formula (I): R 1 , R 3 and R 4 are -H and R 2 is -CH3 ).
  • Specific examples of cyclic analogs comprise a – Abu 4 -S- Ala 7 - or - Ala 4 -S- Ala 7 - linkage.
  • the invention provides an Ang-(1-7) analog with a thioether-bridge between position 4 and position 7 having the amino acid sequence Asp 1 - Arg 2 - Val 3 -Abu 4 -Ile 5 -His 6 -Ala 7 (SEQ ID NO: 15) or the amino acid sequence Asp 1 -Arg 2 - Val 3 -Ala 4 -Ile 5 - His 6 -Ala 7 (SEQ ID NO: 16), which are represented by the following structural diagrams:
  • an Ang analog or derivative of the invention is represented by Formula (V): Xaa 1 -Xaa 2 -Nle 3 -Cyc 4 -Xaa 5 -Xaa 6 -Cyc 7 -Xaa 8 -Xaa 9 -Xaa 10 (V, SEQ ID NO: 17) [0112] As discussed above, one or more of Xaa 1 , Xaa 2 , Xaa 8 , Xaa 9 and Xaa 10 are absent in certain embodiments.
  • Xaa 10 is absent, (2) Xaa 9 and Xaa 10 are absent, (3) Xaa 8 , Xaa 9 and Xaa 10 are absent, (4) Xaa 1 is absent, (5) Xaa 1 and Xaa 10 are absent, (6) Xaa 8 , Xaa 9 and Xaa 10 are absent, (7) Xaa 1 , Xaa 8 , Xaa 9 and Xaa 10 are absent, (8) Xaa 1 and Xaa 2 are absent, (9) Xaa 1 , Xaa 2 and Xaa 10 are absent, (10) Xaa 1 , Xaa 2 , Xaa 9 and Xaa 10 are absent, or (11) Xaa 1 , Xaa 2 , Xaa 8 , Xaa 9 and Xaa 10 are absent.
  • Xaa 1 when present, is any amino acid, but typically a negatively charged amino acid such as Glu or Asp, more typically Asp.
  • Xaa 2 when present, is a positively , charged amino acid such as Arg or Lys, typically Arg.
  • Nle 3 is norleucine.
  • Cyc 4 forms a thioether bridge in conjunction with Cyc 7 .
  • Cyc 4 can be a D-stereoisomer and/or a L-stereoisomer, typically a D-stereoisomer. Examples of Cyc 4 (taken with Cyc 7 ) are shown in Formulas (I), (II) and (III).
  • R groups in Formulae (I), (II) and (III) are —H or —CH, especially —H.
  • Xaa 5 is an aliphatic amino acid, such as Leu, Nle, Ile or Val, typically Ile.
  • Xaa 6 is His.
  • Cyc 7 forms a thioether bridge in conjunction with Cyc 4 , such as in Formula (I), (II) or (III). Cyc 7 can be a D-stereoisomer and/or a L-stereoisomer, typically a L-stereoisomer. Examples of Cyc7 (t ,aken with Cyc) are shown in Formulas (I), (II) and (III).
  • R groups in Formulae (I), (II) and (III) are —H or —CH, especially —H.
  • Xaa 8 when present, is any amino acid other than Pro, typically Phe or Ile. In certain embodiments, Ile results in an inhibitor of Ang (1-8). In certain embodiments, Phe maintains the biological activity of Ang (1-8) or Ang (1-10).
  • Xaa 9 when present, is His.
  • Xaa 10 when present, is an aliphatic residue, for example, Ile, Val or Leu, typically Leu.
  • one or more of Xaa 1 -Xaa 10 is identical to the corresponding amino acid in naturally-occurring Ang (including Ang-(1-7), Ang (1-8), Ang (1-9), Ang (1-10), Ang (2-7), Ang(2-8), Ang (2-9), Ang (2-10), Ang (3-8), Ang (3-9) and Ang(3-10).
  • all but one or two of Xaa 1 -Xaa 10 are identical to the corresponding amino acid in naturally-occurring Ang.
  • Xaa 1 -Xaa 10 are identical to the corresponding amino acid in naturally-occurring Ang.
  • Cyc4 and Cyc7 are independently selected from Abu (2-aminobutyric acid) and Ala (alanine), where Ala is present at least one position.
  • cyclic analogs comprising a thioether linkage formed by-Ala 4 -S- Ala 7 -(Formula (I), where R 1 -R 4 are each —H); -Ala 4 -S-Abu 7 - (Formula (I): R 1 -R 3 are — H and R 4 is —CH3) or –Abu 4 -S-Ala 7 - (Formula (I): R 1 -R 3 and R 4 are —H and R 2 is — CH3).
  • Specific cyclic analogs comprise a-Abu 4 -S-Ala 7 - or-Ala 4 -S-Ala 7 -linkage.
  • the invention provides an Ang-(1-7) analog or derivative with a thioether-bridge between position 4 and position 7 having the amino acid sequence Asp 1 -Arg 2 -Nle 3 -Abu 4 -Ile 5 -His 6 -Ala 7 (SEQ ID NO:18) or the amino acid sequence Asp 1 -Arg 2 -Nle 3 -Ala 4 -Ile 5 -His 6 -Ala 7 (SEQ ID NO:19).
  • the invention provides an Ang-(1-8) analog or derivative with a thioether-bridge between position 4 and position 7 having Ang-(1-8) antagonistic activity, in particular an Ang(1- 8) analog or derivative having the amino acid sequence Asp 1 -Arg 2 -Nle 3 -Abu 4 -Ile 5 -His 6 - Ala 7 -Ile 8 (SEQ ID NO:20), or the amino acid sequence Asp 1 -Arg 2 -Nle 3 -Ala 4 -Ile 5 -His 6 - Ala 7 -Ile 8 (SEQID NO:21).
  • An alkyl group is a straight chained or branched nonaromatic hydrocarbon that is completely saturated.
  • a straight chained or branched alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10.
  • straight chained and branched alkyl groups include methyl, ethyl, n-propyl, iso-propyl. n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl and octyl.
  • a C1-C4 straight chained or branched alkyl group is also referred to as a “lower alkyl” group.
  • An aralkyl group is an alkyl group substituted by an aryl group.
  • Aromatic (aryl) groups include carbocyclic aromatic groups such as phenyl, naphthyl, and anthracyl, and heteroaryl groups such as imidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl pyrrolyl pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl.
  • Aromatic groups also include fused polycyclic aromatic ring systems in which a carbocyclic aromatic ring or heteroaryl ring is fused to one or more other heteroaryl rings.
  • the present invention provides methods of treating lung disease including administering to a subject who is suffering from or susceptible to one or more respiratory complications an angiotensin (1-7) receptor agonist.
  • angiotensin-(1-7) receptor agonist encompasses any molecule that has a positive impact in a function of an angiotensin-(1-7) receptor, in particular, the G- protein coupled Mas receptor.
  • an angiotensin-(1-7) receptor agonist directly or indirectly enhances, strengthens, activates and/or increases an angiotensin-(1-7) receptor (i.e., the Mas receptor) activity.
  • an angiotensin-(1-7) receptor agonist directly interacts with an angiotensin-(1-7) receptor (i.e., the Mas receptor).
  • Such agonists can be peptidic or non-peptidic including, e.g., proteins, chemical compounds, Small molecules, nucleic acids, antibodies, drugs, ligands, or other agents.
  • the angiotensin (1-7) receptor agonist is a non-peptidic agonist.
  • An exemplary class of angiotensin-(1-7) receptor agonists are 1-(p- thienylbenzyl) imidazoles.
  • R 1 is halogen, hydroxyl, (C 1 -C 4 )-alkoxy, (C 1 -C 8 )-alkoxy wherein 1 to 6 carbon atoms are replaced by the heteroatoms O, S, or NH (preferably by O), (C 1 -C 4 )-alkoxy substituted by a saturated cyclic ether such as tetrahydropyran or tetrahydrofuran, O—(C 1 -C 4 )- alkenyl, O (C 1 -C 4 )-alkylaryl, or aryloxy that is unsubstituted or substituted by a substituent selected from halogen, (C-C)-alkyl, (C-C)-alkoxy and trifluoromethyl; R 2 is CHO, COOH, or (3) CO-O (
  • an angiotensin-(1-7) receptor agonist is AVE 0991, 5-formyl-4-methoxy-2-phenyl-14-2-(ethylaminocarbonylsulfonamido)-5-isobutyl-3- thienyl phenyl-methyl-imidazole, which is represented by the following structure:
  • Another exemplary class of angiotensin-(1- 7) receptor agonists are p- thienylbenzylamides. Examples of these non-peptide angiotensin-(1- 7) receptor agonists are represented by Structural Formula (VII):
  • R 1 is (C 1 -C 5 )-alkyl that is unsubstituted or substituted by a radical chosen from NH 2 , halogen, O-(C 1 -C 3 )-alkyl, CO-O-(C 1 -C 3 )-alkyl and CO 2 H, (C 3 -C 8 )-cycloalkyl, (C 1 -C 3 )- alkyl-(C 3 -C 8 )-cycloalkyl, (C 6 -C 10 )-aryl that is unsubstituted or substituted by a radical chosen from halogen and O-(C 1 -C 3 )-alkyl, (C 1 -C 3 )-alkyl-(C 6 -C 10 )-aryl where the aryl radical is unsubstituted or substituted by a radical chosen from halogen and O-(C 1 -C 3 )- alkyl,
  • angiotensin-(1- 7) receptor agonists are described in U.S. Patent No.6,235,766, the contents of which are incorporated by reference herein.
  • Various angiotensin-(1-7) receptor agonists described above can be present as pharmaceutically acceptable salts.
  • a pharmaceutically acceptable salt refers to salts that retain the desired activity of the peptide or equivalent compound, but preferably do not detrimentally affect the activity of the peptide or other component of a system, which uses the peptide.
  • examples of such salts are acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like.
  • Salts may also be formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, and the like.
  • Salts formed from a cationic material may utilize the conjugate base of these inorganic and organic acids.
  • Salts may also be formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel and the like or with an organic cation formed from N,N'- dibenzylethylenediamine or ethylenediamine, or combinations thereof (e.g., a zinc tannate salt).
  • polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel and the like or with an organic cation formed from N,N'- dibenzylethylenediamine or ethylenediamine, or combinations thereof (e.g., a zinc tannate salt).
  • organic cation formed from N,N'- dibenzylethylenediamine or ethylenediamine, or combinations thereof (e.g., a zinc tannate salt).
  • the non-toxic, physiologically acceptable salts are preferred.
  • the salts can be formed by conventional means such as by reacting the free acid or free base forms of the product with one or more equivalents of the appropriate acid or base in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed in vacuo or by freeze-drying, or by exchanging the cations of an existing salt for another cation on a suitable ion exchange resin.
  • An alkyl group is a straight chained or branched non-aromatic hydrocarbon that is completely saturated. Typically, a straight chained or branched alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10.
  • straight chained and branched alkyl groups examples include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl and octyl.
  • a C 1-C4 straight chained or branched alkyl group is also referred to as a "lower alkyl" group.
  • An alkenyl group is a straight chained or branched non-aromatic hydrocarbon that is includes one or more double bonds. Typically, a straight chained or branched alkenyl group has from 2 to about 20 carbon atoms, preferably from 2 to about 10.
  • Aromatic (aryl) groups include carbocyclic aromatic groups such as phenyl, naphthyl, and anthracyl, and heteroaryl groups such as imidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl, pyrrolyl, pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl.
  • Aromatic groups also include fused polycyclic aromatic ring systems in which a carbocyclic aromatic ring or heteroaryl ring is fused to one or more other heteroaryl rings. Examples include benzothienyl, benzofuryl, indolyl, quinolinyl, benzothiazole, benzoxazole, benzimidazole, quinolinyl, isoquinolinyl and isoindolyl. [0129] An aralkyl group is an alkyl group substituted by an aryl group.
  • an Ang-(1-7) peptide o r angiotensin (1-7) receptor agonist as described herein of the invention can be administered to a subject alone (e.g., as a purified peptide or compound), or as a component of a composition or medicament ( e.g., in the manufacture of a medicament for the treatment of the disease), as described herein.
  • the compositions can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition.
  • the carrier and composition can be sterile.
  • the formulation should suit the mode of administration, for example intravenous, subcutaneous or oral administration.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof.
  • the pharmaceutical preparations can, if desired, be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring and/or aromatic substances and the like) which do not deleteriously react with the active compounds or interference with their activity.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring and/or aromatic substances and the like
  • a water-soluble carrier suitable for intravenous administration is used.
  • Ang (1-7) is formulated with cyclodextrin for oral administration as described in, for example, WO 94/20136, the contents of which are incorporated herein by reference.
  • the composition or medicament can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, sustained release formulation, or powder.
  • the composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • the composition or medicament can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings.
  • a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
  • an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • An Ang-(1-7) peptide or angiotensin (1-7) receptor agonist as described herein can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
  • An Ang-(1-7) peptide or angiotensin (1-7) receptor agonist as described herein is administered by any appropriate route.
  • an Ang-(1-7) peptide or angiotensin (1-7) receptor agonist described herein is administered subcutaneously.
  • the term "subcutaneous tissue”, is defined as a layer of loose, irregular connective tissue immediately beneath the skin.
  • the subcutaneous administration may be performed by injecting a composition into areas including, but not limited to, thigh region, abdominal region, gluteal region, or scapular region.
  • an Ang-(1-7) peptide or angiotensin (1-7) receptor agonist described herein is administered intravenously.
  • an Ang- (1-7) peptide or angiotensin (1-7) receptor agonist described herein can be administered by inhalation, parenterally, intradermally, transdermally, rectally, or transmucosally. More than one route can be used concurrently, if desired.
  • a composition is administered in a therapeutically effective amount and/or according to a dosing regimen that is correlated with a particular desired outcome (e.g., with treating or reducing severity of lung disease).
  • Particular doses or amounts to be administered in accordance with the present invention may vary, for example, depending on the nature and/or extent of the desired outcome, on particulars of route and/or timing of administration, and/or on one or more characteristics (e.g., weight, age, personal history, genetic characteristic, lifestyle parameter, severity of pulmonary defect and/or level of risk of pulmonary defect, etc., or combinations thereof). Such doses or amounts can be determined by those of ordinary skill. In some embodiments, an appropriate dose or amount is determined in accordance with standard clinical techniques.
  • an appropriate dose or amount is a dose or amount sufficient to reduce a disease severity index score by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more.
  • an appropriate dose or amount is a dose or amount sufficient to reduce a disease severity index score by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%.
  • an appropriate dose or amount is determined through use of one or more in vitro or in vivo assays to help identify desirable or optimal dosage ranges or amounts to be administered.
  • an Ang-(1-7) peptide or angiotensin (1-7) receptor agonist is administered at a therapeutically effective amount.
  • the term "therapeutically effective amount” is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present invention. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., treating, modulating, curing, preventing and/or ameliorating the underlying disease or condition).
  • appropriate doses or amounts to be administered may be extrapolated from dose- response curves derived from in vitro or animal model test systems.
  • Therapeutically effective dosage amounts of angiotensin (1-7) peptides or angiotensin (1-7) receptor agonists, including derivatives, analogs, and/or salts may be present in varying amounts in various embodiments.
  • a therapeutically effective amount of an angiotensin (1-7) peptide may be an amount ranging from about 10-1000 mg (e.g., about 20 mg - 1,000 mg, 30 mg - 1,000 mg, 40 mg - 1,000 mg, 50 mg - 1,000 mg, 60 mg - 1,000 mg, 70 mg - 1,000 mg, 80 mg - 1,000 mg, 90 mg - 1,000 mg, about 10-900 mg, 10-800 mg, 10-700 mg, 10- 600 mg, 10-500 mg, 100-1000 mg, 100-900 mg, 100-800 mg, 100-700 mg, 100-600 mg, 100-500 mg, 100-400 mg, 100-300 mg, 200-1000 mg, 200-900 mg, 200-800 mg, 200- 700 mg, 200-600 mg, 200-500 mg, 200-400 mg, 300-1000 mg, 300-900 mg, 300-800 mg, 300-700 mg, 300-600 mg, 300-500 mg, 400 mg, 300-1000 mg, 300-900 mg, 300-800 mg, 300-700 mg, 300-600 mg, 300-500 mg, 400 mg
  • an angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist is present in an amount of or greater than about 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg.
  • an angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist is present in an amount of or less than about 1000 mg, 950 mg, 900 mg, 850 mg, 800 mg, 750 mg, 700 mg, 650 mg, 600 mg, 550 mg, 500 mg, 450 mg, 400 mg, 350 mg, 300 mg, 250 mg, 200 mg, 150 mg, or 100 mg.
  • the therapeutically effective amount described herein is provided in one dose. In some embodiments, the therapeutically effective amount described herein is provided in one day.
  • a therapeutically effective dosage amount may be, for example, about 0.001 mg/kg weight to 500 mg/kg weight, e.g., from about 0.001 mg/kg weight to 400 mg/kg weight, from about 0.001 mg/kg weight to 300 mg/kg weight, from about 0.001 mg/kg weight to 200 mg/kg weight, from about 0.001 mg/kg weight to 100 mg/kg weight, from about 0.001 mg/kg weight to 90 mg/kg weight, from about 0.001 mg/kg weight to 80 mg/kg weight, from about 0.001 mg/kg weight to 70 mg/kg weight, from about 0.001 mg/kg weight to 60 mg/kg weight, from about 0.001 mg/kg weight to 50 mg/kg weight, from about 0.001 mg/kg weight to 40 mg/kg weight, from about 0.001 mg/kg weight to 30 mg/kg weight, from about 0.001 mg/kg weight to 25 mg/kg weight, from about 0.001 mg/kg weight to 20 mg/kg weight, from about 0.00 0.001 mg/kg weight to
  • a therapeutically effective dosage amount may be, for example, about 0.001 mg/kg weight to about 1 mg/kg weight, e.g.
  • a therapeutically effective dosage amount may be, for example, about 0.0001 mg/kg weight to 0.1 mg/kg weight, e.g.
  • the therapeutically effective dose may be 0.0001 mg/kg weight, 0.0002 mg/kg weight, 0.0003 mg/kg weight, 0.0004 mg/kg weight, 0.0005 mg/kg weight, 0.0006 mg/kg weight, 0.0007 mg/kg weight, 0.0008 mg/kg weight, 0.0009 mg/kg weight, 0.001 mg/kg weight, 0.002 mg/kg weight, 0.003 mg/kg weight, 0.004 mg/kg weight, 0.005 mg/kg weight, 0.006 mg/kg weight, 0.007 mg/kg weight, 0.008 mg/kg weight, 0.009 mg/kg weight, 0.01 mg/kg weight, 0.02 mg/kg weight, 0.03 mg/kg weight, 0.04 mg/kg weight, 0.05 mg/kg weight, 0.06 mg/kg weight, 0.07 mg/kg weight, 0.08 mg/kg weight, 0.09 mg/kg weight, or 0.1 mg/kg weight.
  • the angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-1,000 ⁇ g/kg/day (e.g., ranging from about 1-900 ⁇ g/kg/day, 1-800 ⁇ g/kg/day, 1-700 ⁇ g/kg/day, 1-600 ⁇ g/kg/day, 1-500 ⁇ g/kg/day, 1- 400 ⁇ g/kg/day, 1-300 ⁇ g/kg/day, 1-200 ⁇ g/kg/day, 1-100 ⁇ g/kg/day, 1-90 ⁇ g/kg/day, 1- 80 ⁇ g/kg/day, 1-70 ⁇ g/kg/day, 1-60 ⁇ g/kg/day, 1-50 ⁇ g/kg/day, 1-40 ⁇ g/kg/day, 1-30 ⁇ g/kg/day, 1-20 ⁇ g/kg/day, 1-10 ⁇
  • the angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-500 ⁇ g/kg/day. In some embodiments, the angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-100 ⁇ g/kg/day. In some embodiments, the angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-60 ⁇ g/kg/day.
  • the angiotensin (1-7) peptide is administered at an effective dose selected from about 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 ⁇ g/kg/day.
  • Dosing Schedules [0144] Various embodiments may include differing dosing regimen.
  • the angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist is administered via continuous infusion.
  • the continuous infusion is intravenous. In other embodiments, the continuous infusion is subcutaneous.
  • the angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist is administered bimonthly, monthly, twice monthly, triweekly, biweekly, weekly, twice weekly, thrice weekly, daily, twice daily, or on another clinically desirable dosing schedule.
  • the dosing regimen for a single subject need not be at a fixed interval, but can be varied over time, depending on the needs of the subject.
  • Kits [0145]
  • the present invention further provides kits or other articles of manufacture which contains an Ang-(1-7) peptide, an angiotensin (1-7) receptor agonist or a formulation containing the same and provides instructions for its reconstitution (if lyophilized) and/or use.
  • Kits or other articles of manufacture may include a container, a syringe, vial and any other articles, devices or equipment useful in administration (e.g., subcutaneous, by inhalation).
  • Suitable containers include, for example, bottles, vials, syringes (e.g., pre-filled syringes), ampules, cartridges, reservoirs, or lyo-jects.
  • the container may be formed from a variety of materials such as glass or plastic.
  • a container is a pre-filled syringe.
  • Suitable pre-filled syringes include, but are not limited to, borosilicate glass syringes with baked silicone coating, borosilicate glass syringes with sprayed silicone, or plastic resin syringes without silicone.
  • the container may hold formulations and a label on, or associated with, the container that may indicate directions for reconstitution and/or use.
  • the label may indicate that the formulation is reconstituted to concentrations as described above.
  • the label may further indicate that the formulation is useful or intended for, for example, subcutaneous administration.
  • a container may contain a single dose of a stable formulation containing an Ang-(1-7) peptide or angiotensin (1-7) receptor agonist.
  • a single dose of the stable formulation is present in a volume of less than about 15 ml, 10 ml, 5.0 ml, 4.0 ml, 3.5 ml, 3.0 ml, 2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, or 0.5 ml.
  • a container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the formulation.
  • Kits or other articles of manufacture may further include a second container comprising a suitable diluent (e.g.,BWFI, saline, buffered saline).
  • a suitable diluent e.g.,BWFI, saline, buffered saline.
  • the final protein concentration in the reconstituted formulation will generally be at least 1 mg/ml (e.g., at least 5 mg/ml, at least 10 mg/ml, at least 20 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/ml).
  • Kits or other articles of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, kits or other articles of manufacture may include an instruction for self-administration.
  • Example 1 Correlation between serum levels of Ang-(1-7) and disease progression in coronavirus infection
  • This example describes measurement of serum levels of Ang-(1-7) peptide in the serum of individuals suffering from lung disease, for example, as a complication of coronavirus infection, and evaluating the correlation between Ang-(1-7) levels and disease progression.
  • Serum samples are collected from healthy individuals, and individuals infected with coronavirus, for example, during early stage of infection (i.e. within 2 weeks of onset of symptoms) and during late stage of infection (i.e. after 2 weeks of onset of symptoms).
  • Levels of Ang-(1-7) are measured in the samples collected using enzyme immunoassays.
  • the serum levels of Ang-(1-7) are compared between healthy individuals and individuals infected with coronavirus, if Ang-(1-7) levels are decreased in coronavirus infected individuals, it indicates downregulation of the ACE2 receptor and the alternative renin-angiotensin system.
  • Serum Ang-(1-7) levels are correlated with clinical disease progression and symptom severity through scoring clinical parameters such as fever, cough, and lung function tests including but not limited to spirometry and lung volume tests, pulse oximetry and chest imaging. Comparisons between early stage and late stage infections is made.
  • Ang-(1-7) When high levels of Ang-(1-7) in serum are correlated with slower clinical disease progression and reduced symptom severity, it indicates that the proposed administration of Ang-(1-7) will have therapeutic efficacy.
  • TXA127 for the Treatment of Pneumonia Secondary to Infection by Coronavirus COVID-19 [0157] Infection by the coronavirus, COVID-19, is a worldwide epidemic, the future course of which is unknown at this time.
  • TXA127 which is a pharmaceutical formulation of the naturally occurring human peptide angiotensin-(1-7) (ANG-(1-7)). It is contemplated that TXA127 is the principal agonist in the Alternative Renin Angiotensin System (ARAS) pathway.
  • ARAS Renin Angiotensin System
  • ARAS counteracts the effects of the “Classical” pathway in the Renin Angiotensin System (RAS) and the balance of these two pathways is important in determining the outcome in various diseases. See Figure 1.
  • the rationale for the use of TXA127 in treating COVID-19 pneumonia has at least four components: 1.
  • the binding site for COVID-19 is the cell membrane bound enzyme ACE2, which is expressed in many tissues.
  • ACE2 is also the attachment site for SARS and avian flu H5N1.
  • Binding of COVID-19 to ACE2 downregulates the expression of the enzyme.
  • Downregulation of ACE2 will reduce the amount of Ang-(1-7) and alter the balance between the Classical and Alternative pathways of the RAS with detrimental effects. 4.
  • Treatment with TXA127 will restore the balance and lead to a reduction in severity of disease and mortality.
  • Clinical study using TXA127 to treat pneumonia secondary to infection by coronavirus COVID-19 is carried out as follows: [0161] Approximately 100 patients with COVID-19 pneumonia are enrolled in the study. Phase 1 trial recruiting approximately 20 patients designed to confirm safety and inform the design of a Phase 2 trial. Implementation of a double blind, placebo-controlled Phase 2 trial powered to show efficacy. Implementation of a Phase 3 trial leading to regulatory approval. [0162] Subcutaneous and/or intravenous administration of TXA127 is used to treat hospitalized patients. Oral administration is used for treatment outpatients with respiratory symptoms, which prevents their progressing to hospitalization.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Pulmonology (AREA)
  • Vascular Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
EP21715458.2A 2020-03-13 2021-03-12 Verfahren und zusammensetzungen zur behandlung von coronavirus-infektionen Withdrawn EP4117703A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202062989145P 2020-03-13 2020-03-13
PCT/US2021/022077 WO2021183863A1 (en) 2020-03-13 2021-03-12 Methods and compositions for treatment of coronavirus infection

Publications (1)

Publication Number Publication Date
EP4117703A1 true EP4117703A1 (de) 2023-01-18

Family

ID=75278436

Family Applications (1)

Application Number Title Priority Date Filing Date
EP21715458.2A Withdrawn EP4117703A1 (de) 2020-03-13 2021-03-12 Verfahren und zusammensetzungen zur behandlung von coronavirus-infektionen

Country Status (3)

Country Link
US (1) US20230110554A1 (de)
EP (1) EP4117703A1 (de)
WO (1) WO2021183863A1 (de)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022094025A1 (en) * 2020-10-30 2022-05-05 Gere Dizerega Angiotensin-1-7 for the treatment of covid-19
EP4220172A1 (de) * 2022-01-27 2023-08-02 Université Libre de Bruxelles Covid-19-theranostikum
EP4371556A1 (de) 2022-11-15 2024-05-22 Explicat Pharma GmbH Pharmazeutische angiotensin(1-7)-zusammensetzungen zur inhalation

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4244946A (en) 1979-06-11 1981-01-13 The Salk Institute For Biological Studies Water-soluble peptides affecting gonadal function
US4305872A (en) 1979-10-19 1981-12-15 Kenneth Wingrove Polypeptide derivatives
US4316891A (en) 1980-06-14 1982-02-23 The Salk Institute For Biological Studies Extended N-terminal somatostatin
CA2134753A1 (en) 1993-03-08 1994-09-15 Peter K. Chiang Cyclodextrin-peptide compositions
US20020168761A1 (en) 2000-01-24 2002-11-14 Gour Barbara J. Peptidomimetic modulators of cell adhesion
US6031072A (en) 1996-07-12 2000-02-29 Mcgill University Compounds and methods for modulating cell adhesion
YU78601A (sh) 1999-05-05 2005-07-19 Aventis Pharma Deutschland Gmbh. 1-(p-tienilbenzil)-imidazoli kao agonisti angiotenzin-(1-7)- receptora, postupak za njihovu proizvodnju, njihova primena i farmaceutski preparati koji ih sadrže
EP2163259B1 (de) * 2008-09-12 2012-06-13 Charité-Universitätsmedizin Berlin (Charité) Verwendung eines Ang-(1-7)-Rezeptorantagonisten bei akuter Lungenverletzung

Also Published As

Publication number Publication date
US20230110554A1 (en) 2023-04-13
WO2021183863A1 (en) 2021-09-16

Similar Documents

Publication Publication Date Title
US20230110554A1 (en) Methods and compositions for treatment of coronavirus infection
US10960045B2 (en) Methods and compositions for the treatment of epidermolysis bullosa
EP2903605B1 (de) Angiotensin zur behandlung von hirnzuständen
AU2013216910A1 (en) Compositions and methods for treatment of peripheral vascular disease
US20180050079A1 (en) Angiotensin peptides in treating marfan syndrome and related disorders
US8557958B1 (en) Compositions and methods for treatment of diabetes
CA2940751C (en) Uses of an angiotensin (1-7) peptide for the delayed treatment of stroke
US20160199436A1 (en) Methods and compositions for the treatment of amyotrophic lateral sclerosis
AU2014332346B2 (en) Novel peptide compositions
WO2015057403A2 (en) Compositions and methods for treatment of inflammatory bowel disease
WO2017218623A1 (en) Methods and compositions for the treatment of epidermolysis bullosa
AU2014269028A1 (en) Angiotensins in muscular dystrophy
US20160296591A1 (en) Compositions and methods for treatment of bdnf-related conditions
US9133241B2 (en) Peptide compositions

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20221011

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20231003